To evaluate the newly developed next generation sequencing assay by analyzing patients with isolated eye abnormalities collected in earlier studies including patients enrolled through eyeGENE program: This project began several years ago in the OGFVB/NEI. We first developed a screening tool by the resequencing CHIP on 93 genes. Later, we developed another approach using microdroplet PCR technology and the next generation sequencing (NGS) to analyze 184 genes. Our manuscript of the microdroplet PCR technology has been submitted. Later on, we adapted a target capture plus NGS technology. We developed a new panel composed of 330 genes related to retinal function and development. Using NGS technology, we have been more actively analyzing patient with variety categories of retinal dystrophies in a more efficient and accurate approach. We have performed NGS sequencing for more than 350 samples with a variety of ophthalmic diseases. We are continuing the procedures to analyze more patients with RP. Meanwhile, we have built our own in house bioinformatics pipeline to analyze NGS data. We are capable to analyze all kinds of NGS data, including WES data. Using this pipeline, we have built our own bioinformatics pipeline and have analyzed NGS data from different projects. To date, we have analyzed over 300 samples for several projects including the following: o Perform analysis for NISC project. Assisted in the development of the pipeline of 100 samples to be analyzed from NISC. o Perform analysis for whole exome sequencing project (WES). Analyzed samples and developed protocol for whole exome sequencing analysis. We also used these samples to search for ACMG common genes. We have also purchased a software package for mutation analysis, Alamut. This software assists us in the determination of variant pathogenicity. Alamut consolidates data from 3 mutation algorithms, population frequencies, nucleotide and amino acid conservation, protein structures, and splicing information into one platform. This saves the DDL lab significant time and effort when analyzing variants. We created a custom DDL Variant Report to use with Alamut for variant analysis. The report provides easy access to variant information without the need for special software. o Improved overall sequencing quality. Initially, we loaded our sequencing runs at 20pM. As of now, we are loading all of our sequencing runs at 10pM. Doing so increases the number of reads per sample while improving the amount of coverage that is received for each sample. The overall quality of the runs has improved as well; specifically, Q30 scores and Cluster Pass Filtering Scores. o Continuously working to develop all Standard Operating Procedures in accordance with CLIA regulations, so the Next-Generation sequencing protocol can become clinical procedure. Other projects: To develope a RNA/cDNA Sure Select Targeted Enrichment project. Starting a project with Agilent for Strand-specific RNA library preparation and target enrichment. o Determining the number of samples and parameters needed for optimal clustering o Adjusting the loading concentration of the sequencing run based on our sequencing needs o Pooling the samples together at specified concentration(10nM) o Manually correcting index sequences o Adjusting the sequencing parameters: Read1:151 cycles, Read2:151, Index:8bp o Perform analysis and data processing Through our successful sequencing, we are now attempting to sequence the entire cDNA library using the same parameters: Although, additional testing and troubleshooting will be needed in order to complete this project. Proposed a new method to develop a new sequencing platform covering all genes known to cause inherited eye diseases by deep sequencing. We are currently testing a new method provided by Agilent Technologies that will allow us to provide a superior method for comprehensive and efficient targeted sequencing that offers cost efficiencies and a streamlined workflow; while, accelerating the turn-around time from sample to date and providing deep coverage of genomic regions of interest. This project will allow us to potentially decrease the turnaround time frame <2 weeks /16 samples along with the cost per sample to $284. So far, we have processed a total of 36 samples using this method. New approach to perform NGS sequencing We are currently testing a new method provided by Agilent Technologies that will allow us to provide a superior method for comprehensive and efficient targeted sequencing that offers cost efficiencies and a streamlined workflow; while, accelerating the turn-around time from sample to date and providing deep coverage of genomic regions of interest. This project will allow us to potentially decrease the turnaround time frame <2 weeks /16 samples along with the cost per sample to $284. Clinical laboratory for FDA approved clinical trial, XLRS2 project We are the clinical laboratory for the XLRS2 clinical trial by processed 77 Serum samples, 9 AAV Vector Dilution Storage, 3 FDA site visits, and 1 shipment for clinical analysis to the Advanced Bioscience Laboratories, Inc (ABL) in support of XLRS2 clinical trial. It included performing 77 serum extractions on nine participants and stored 9 AAV vector dilutions. Time spent processing each serum sample is approximately 45 minutes to 1 hour. Preparing publications for current collaborative research DDL commits to publish our research and our collaborative research results. Here is the list of our currently manuscripts in different stages: 1. Xiaodong Jiao Anren Li, Zi-Bing Jin, Xinjing Wang, Alessandro Iannaccone, Elias Traboulsi, Michael B. Gorin, Francesca Simonelli, and J. Fielding Hejtmancik* Identification and Population History of CYP4V2 mutations in Patients with Bietti Crystalline Corneoretinal Dystrophy (post peer-review resubmission) 2. Annalisa Nicoletti, Lucia Ziccardi, Paolo Enrico Maltese, Sabrina Benedetti, Orazio Palumbo, Michelina Rendina, Leonardo DAgruma, Benedetto Falsini, Xinjing Wang, Matteo Bertelli*. Design and validation of a new MLPA-based assay for the detection of RS1 gene deletions and application in a large family with X-linked juvenile Retinoschisis (post peer-review revision) 3. Jackson Gao, Leera DSounza, Keith Wetherby, Christian Antolik, Melissa Reeves, David Adams, Xinjing Wang* Retrospective Mutation Analysis in Oculocutaneous Albinism Patients for a 2.7 kb deletion mutation in the OCA2 gene revealed a co-segregation of the controversial allele, p.R305W (post peer-review revision). 4. Xinjing Wang*, Wadih Zein, Leera DSouza, Chimere Roberson, Keith Wetherby, Hong He, Angela Villarta, Delphine Blaine, Amy Turriff, Kory Johnson, Yang C Fann. Applying the next generation sequencing by microdroplet PCR to determine the disease causing mutations in retinal dystrophies (submitted). 5. Xinjing Wang*, Danyao Nie, Chimere Roberson, Keith Wetherby, Angela Villarta, Kerry Goetz, Alexandra Garafalo, Rebecca Parrish, Santa Tumminia, Kory Johnson, Yang C Fann. NGS Analysis of Corneal Dystrophy Related Genes Using Modified Microdroplet PCR Protocol in Patients with Corneal Dystrophies (manuscript in preparation). 6. Xinjing Wang*, Chimere Roberson, Keith Wetherby, Melissa Reeves, Wadih Zein, Delphine Blain, Amy Turriff, Robert Hufnagel, Santa J. Tumminia, Brian Brook. NGS analysis of 100 samples from the eyeGENE program to determine the disease causing mutations in retinal dystrophies (manuscript in preparation).